The long term goal of this proposal is to elucidate the protease signaling pathways involved in innate immunity. Specifically we propose to identify the proteins that are cleaved during the innate immune response, and to identify the proteases responsible for the cleavage event. Activation of proteases, notably caspases-1 and -5, is a hallmark of the innate immune response, yet only a few proteins are known to be processed, including some very important pro-inflammatory cytokines IL-1(3, IL-18 and IL-33. From our work (unpublished) and that of others, there are now known to be >300 proteins cleaved by caspases during apoptosis. The inflammatory caspases (-1 and -5) have very similar catalytic efficiencies compared to their apoptotic cousins (caspase-3, -7, -8 and -9). The substrate specificities of the inflammatory caspases do not appear more restricted than the apoptotic caspases. What accounts for the 100-fold difference in the apparent number of proteins known to be processed by these two families? We present two hypotheses and tests for them. First, there may be many more proteins cleaved by the inflammatory caspases than are currently known. We will address this using a new and general mass spectrometry-based method for global profiling of proteolysis in cells (we call "degradomics"). The Wells lab has engineered an enzyme, called subtiligase, that selectively ligates biotinylated peptides onto the ?-amines and not ?-amines of proteins allowing us to purify and sequence newly cleaved proteins. Preliminary studies in apoptotic Jurkat cells have already identified 183 aspartate cleaved proteins, of which 151 are unreported caspase substrates. We propose a combination of cellular ("forward") and in vitro ("reverse") degradomics experiments to provide the most comprehensive map yet of proteins cleaved during an innate immune stimulus, and the protease(s) responsible. A second possibility is that the inflammatory caspases cleave proteins that are sequestered in secretory vesicles or extracellular compartments. This we can test by applying our degradomics methodology to cell fractions to specifically look at proteins secreted during innate immune stimuli. Relevance to public health: We believe our studies will reveal new pro-inflammatory proteins and the proteases that process them. Ultimately, the activity of these pro-inflammatory proteins or their processing proteases can be blocked to provide new therapeutics for inflammatory diseases.